Methods

Mesenchymal stem cells were harvested from the femora of young, adult, and osteopenic Wistar rats. Cluster of differentiation (CD) marker and CXCR-4 expression was measured using flow cytometry. Cellular proliferation was measured using Alamar Blue, osteogenic differentiation was measured using alkaline phosphatase expression and alizarin red production, and adipogenic differentiation was measured using Oil red O. Cells were incubated in Boyden chambers to quantify their migration towards SDF-1. Data was analyzed using a Student’s t-test, where p-values < 0.05 were considered significant.

Methods

Dexamethasone, diclofenac, and decorin, in different concentrations, were employed to treat fibroblasts from arthrofibrotic tissue (AFib). Cell proliferation was measured by DNA quantitation, and viability was analyzed by Live/Dead staining. The levels of procollagen type I N-terminal propeptide (PINP) and procollagen type III N-terminal propeptide (PIIINP) were evaluated with enzyme-linked immunosorbent assay (ELISA) kits. In addition, the expressions of fibrotic markers were detected by real-time polymerase chain reaction (PCR). Fibroblasts isolated from healthy tissue (Fib) served as control. Further, a rabbit model of joint contracture was used to evaluate the antifibrotic effect of the three different agents.

Methods

The metatarsophalangeal joints of 12 fresh cadaveric bovine feet were skinned and dissected aseptically, and cultured for up to four weeks. Dynamic movement was applied using a custom-made machine on six joints, with the others cultured under static conditions. Chondrocyte viability and matrix glycosaminoglycan (GAG) content were evaluated by the cell viability probes, 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI), and dimethylmethylene blue (DMMB) assay, respectively.

Patients and Methods

Conventional radiological examination of 95 skeletally mature patients with NPS was performed. Patellar morphology was classified according to the Wiberg classification as modified by Baumgartl and Ficat criteria, and trochlear shape was classified according to the Dejour classification.

Methods

Acetabular coverage of all subjects was evaluated using radiological findings (Sharp angle, centre-edge (CE) angle, acetabular roof obliquity (ARO) angle, and minimum joint space width). Genomic DNA was extracted from peripheral blood leukocytes. Agilent’s region-targeted high-density oligonucleotide tiling microarray was used to analyse 64 female AD patients and 32 female control subjects. All statistical analyses were performed using EZR software (Fisher’s exact probability test, Pearson’s correlation test, and Student’s t-test).

Methods

T2DM rats were obtained from the UC Davis group and compared with control Lewis rats. The diabetic rats were sacrificed at ages from six to 12 months, while control rats were sacrificed at six months only. Osteoarthritis severity was determined via histology in four knee quadrants using the OARSI scoring guide. Immunohistochemical staining was also performed as a secondary form of osteoarthritic analysis.

Methods

Human PBMNCs were cultured for seven days with the QQ-culture method using a serum-free medium containing five specific cytokines and growth factors. The QQ-cultured PBMNCs (QQMNCs) obtained were counted and characterised by flow cytometry and real-time polymerase chain reaction (RT-PCR). Angiogenic and osteo-inductive potentials were evaluated using tube formation assays and co-culture with mesenchymal stem cells with osteo-inductive medium in vitro. In order to evaluate the therapeutic potential of QQMNCs, cells were transplanted into an immunodeficient rat femur nonunion model. The rats were randomised into three groups: control; PBMNCs; and QQMNCs. The fracture healing was evaluated radiographically and histologically.

Methods

Human cartilage cells (C28/I2) were transfected with miR-320a or antisense oligonucleotides (ASO)-miR-320a, and treated with IL-1β. Subsequently the expression of collagen type II alpha 1 (Col2α1) and aggrecan (ACAN), and the concentrations of sulfated glycosaminoglycans (sGAG) and matrix metallopeptidase 13 (MMP-13), were assessed. Luciferase reporter assay, qRT-PCR, and Western blot were performed to explore whether pre-B-cell leukemia Homeobox 3 (PBX3) was a target of miR-320a. Furthermore, cells were co-transfected with miR-320a and PBX3 expressing vector, or cells were transfected with miR-320a and treated with a nuclear factor kappa B (NF-κB) antagonist MG132. The changes in Col2α1 and ACAN expression, and in sGAG and MMP-13 concentrations, were measured again. Statistical comparisons were made between two groups by using the two-tailed paired t-test.

Methods

We examined the expression levels of E3 ligases and NF-κB members in callus samples during bone fracture repair by quantitative polymerase chain reaction (qPCR) and the total amount of ubiquitinated proteins by Western blot analysis in wild-type (WT) mice. The expression levels of osteoblast-associated genes in fracture callus from Itch knockout (KO) mice and their WT littermates were examined by qPCR. The effect of NF-κB on Itch expression in C2C12 osteoblast cells was determined by a chromatin immunoprecipitation (ChIP) assay.

Patients and Methods

A single institution retrospective review of all patients presenting with OFD, OFD-like adamantinoma and adamantinoma between 1973 and 2012 was undertaken. Complete data were available for 73 patients (42 with OFD; ten with an OFD-like adamantinoma and 21 with an adamantinoma). The mean follow-up was 10.3 years (3 to 25) for OFD, 9.2 years (3.0 to 26.3) for OFD-like and 11.6 years (0.25 to 33) for adamantinoma.

Methods

MSCs from rabbits were cultured in a control medium and medium with G-CSF (low-dose: 4 μg, high-dose: 40 μg). At one, three, and five days after culturing, cells were counted. Differential potential of cultured cells were examined by stimulating them with a osteogenic, adipogenic and chondrogenic medium.

A total of 30 rabbits were divided into three groups. The low-dose group (n = 10) received 10 μg/kg of G-CSF daily, the high-dose group (n = 10) received 50 μg/kg daily by subcutaneous injection for three days prior to creating cartilage defects. The control group (n = 10) was administered saline for three days. At 48 hours after the first injection, a 5.2 mm diameter cylindrical osteochondral defect was created in the femoral trochlea. At four and 12 weeks post-operatively, repaired tissue was evaluated macroscopically and microscopically.

Methods

Specifically, canine flexor digitorum profundus tendons were used for this study and were divided into the following groups: (1) untreated, unprocessed normal tendon; (2) decellularised tendon; (3) bone marrow stromal cell (BMSC)-seeded tendon; and (4) BMSC-seeded and cyclically stretched tendon. Lateral slits were introduced on the tendon to facilitate cell seeding. Tendons from all four study groups were distracted by a servohydraulic testing machine. Tensile force and displacement data were continuously recorded at a sample rate of 20 Hz until 200 Newton of force was reached. Before testing, the cross-sectional dimensions of each tendon were measured with a digital caliper. Young’s modulus was calculated from the slope of the linear region of the stress-strain curve. The BMSCs were labeled for histological and cell viability evaluation on the decellularized tendon scaffold under a confocal microscope. Gene expression levels of selected extracellular matrix tendon growth factor genes were measured. Results were reported as mean ± SD and data was analyzed with one-way ANOVAs followed by Tukey’s post hoc multiple-comparison test.

Methods

Cartilage defects were created in the load-bearing region of the lateral femoral condyle of mini-type pigs. The defects were repaired with traditional tissue-engineered cartilage, tissue-engineered cartilage constructed with the biotin-avidin (BA) technique, tissue-engineered cartilage constructed with the CBA technique and with autologous cartilage. The biomechanical properties, Western blot assay, histological findings and immunohistochemical staining were explored.

Materials and Methods

A total of 33 adult humeri were used from a previous study where we quantified bone mineral density of the proximal humerus using radiographs and dual-energy x-ray absorptiometry (DEXA), and regional mean cortical thickness and cortical index using radiographs. The bones were fractured in a simulated backwards fall with the humeral head loaded at 2 mm/second via a frustum angled at 30° from the long axis of the bone. Correlations were assessed with ultimate fracture load and these new parameters: cortical index expressed in areas (“areal cortical index”) of larger regions of the diaphysis; the canal-to-calcar ratio used analogous to its application in proximal femurs; and the recently described medial cortical ratio.

Methods

Rotator cuff-derived cells were seeded under inflammatory stimulation conditions (with serum-free medium with 1 ng/ml IL-1ß for three hours), and then cultured in different media: serum-free (control group), serum-free + TA (0.1mg/ml) (TA group), serum-free + 10% PRP (PRP group), and serum-free + TA (0.1mg/ml) + 10% PRP (TA+PRP group). Cell morphology, cell viability, and expression of inflammatory and degenerative mediators were assessed.

Materials and Methods

Human articular cartilage was obtained from patients with and without OA, and chondrocytes were isolated and stimulated by IL-1β. The expression levels of miR-138-5p in cartilage and chondrocytes were both determined. After transfection with miR-138-5p mimics, allele-specific oligonucleotide (ASO)-miR-138-5p, or their negative controls, the messenger RNA (mRNA) levels of aggrecan (ACAN), collagen type II and alpha 1 (COL2A1), the protein levels of glycosaminoglycans (GAGs), and both the mRNA and protein levels of matrix metalloproteinase (MMP)-13 were evaluated. Luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot were performed to explore whether Forkhead Box C1 (FOCX1) was a target of miR-138-5p. Further, we co-transfected OA chondrocytes with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 and then stimulated with IL-1β to determine whether miR-138-5p-mediated IL-1β-induced cartilage matrix degradation resulted from targeting FOXC1.

Patients and Methods

Samples of tissue were taken during 35 two-stage revision arthroplasties (nine total hip and 26 total knee arthroplasties) in patients whose mean age was 67 years (44 to 85). Fourier transform infrared microscopy was used to confirm the presence of PMMA particles. Histomorphometry was performed using Sudan Red and Haematoxylin-Eosin staining. CD-68, CD-20, CD-11(c), CD-3 and IL-17 antibodies were used to immunophenotype the inflammatory cells. All slides were scored semi-quantitatively using the modified Willert scoring system.